Library Construction and RNA Sequencing
Total RNAs was extracted from the root samples of four soybean genotypes under control and stress conditions using TRIzol reagent by following the manufacturer’s protocols (Invitrogen, Carlsbad, CA, USA). The RNA quantity and integrity was checked using Nano-Drop Spectrometer (ND-1000 Spectrophotometer, Thermo Scientific) and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US), respectively. To select the best quality RNA an integrity value ≥7 was used for library construction. Total RNA of 2 μg from each sample was used for the construction of paired-end libraries using Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), in accordance with the manufacturer’s instructions. Qubit® 2.0Fluorometer (Life Technologies, USA) was used to quantify purified libraries, and are authenticated by Agilent 2100-bioanalyzer (Agilent Technologies, USA) to validate the fragment size and concentration. Furthermore, libraries were diluted up to 10 Pm for cBot clustering and RNA-Seq was performed using Illumina HiSeq 2500 platform by Shanghai Biotechnology Corporation.